Lyme Disease (LD) is the most common vector-borne infectious disease in the United States and remains a significant public health concern. The laboratory diagnosis of LD depends on the demonstration of antibodies against Borrelia burgdorferi. Current serologic assays are not specific enough and not sensitive in early LD. New assays are needed and we plan to fill this unmet need. Our main objective is to produce new sensitive and specific peptide based assays for the serodiagnosis of LD. In Phase I we made excellent progress in defining components for a new serologic assay: we developed IR6 peptides with a broader ability to detect antibody reactivity among patients infected with spirochetes expressing different VlsE sequences;of the 17 known US OspC groups we have identified four (B, F, I, and K) as potential targets for epitope mapping;and we evaluated the ability of a multi-antigenic peptide comprised of sequences from OspC (10 residues), FlaB (13 residues) and VlsE-IR6 (17 residues) to bind antibody from individuals with LD. In this Phase II proposal, we will further optimize the peptides containing epitopes from OspC, FlaB and IR6 and will validate new immunoassays in both ELISA and rapid lateral flow point-of-care (POC) formats. An assay with greater specificity and improved sensitivity for detection of B. burgdorferi antibodies in the earliest stages of the disease, could lead to the development of a single-tier test capable of replacing the currently recommended two-tier paradigm. More importantly, given that early diagnosis and treatment of LD prevents illness progression and sequellae, the development of this test will make an important contribution to improvement of human health. PUBLIC HEALTH RELEVANCE: Lyme disease, the most common vector borne infectious disease in the United States affects multiple organ systems. Although prompt diagnosis and treatment prevents or limits serious injury to the systems affected, current sero- diagnostics for Lyme disease lack sensitivity in early disease and are not specific enough to be used alone. Our objective is to replace today's assays with a new multi-antigen peptide test. Because of greater specificity and improvement to the assay's sensitivity, this approach could lead to the development of a single tier assay to detect antibodies against any of the three pathogenic genospecies ofB. burgdorferi in both early and late Lyme disease.